We are now in the process of investigating a series of protein-derived radicals formed by myoglobin, hemoglobin and superoxide dismutase that utilize our new immunological/spin-trapping techniques. Polyclonal antibodies bind specifically to the spin trap/protein adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The ESR signal of the myoglobin-DMPO radical decays within minutes, however a persistent adduct can be detected using mass spectroscopy. This adduct can be visualized using Western blot techniques at a sensitivity 10-fold greater than ESR. Western blot analysis demonstrates that myoglobin specifically forms a protein-derived radical in rat heart supernatant. For the first time, we were able to demonstrate the formation of free radicals inside a cell. When red blood cells were exposed to 50-micromolar hydrogen peroxide, hemoglobin-derived radicals were detected inside the red blood cells. The sites of free radical formation on hemoglobin have been determined by the mass spectroscopy group who discovered a novel histidine free radical.[unreadable] With immuno-spin trapping method we have detected totally unsuspected protein radicals. For instance when LPO was reacted with glutathione (GSH) in the presence of DMPO, we detected an LPO radical-derived DMPO nitrone adduct with ELISA and Western blot. These results suggest that the GSH-dependent LPO radical formation is mediated by the glutathiyl radical, possibly via the reaction of the glutathiyl radical with the heme of compound II to form a heme-centered radical trapped by DMPO. The sensitivity of immuno-spin trapping enables the investigation of low abundance proteins from expression systems. For instance, neuroglobin (Ngb) is a recently discovered protein that belongs to the globin family of proteins. A particularly high Ngb concentration has been reported in the mammalian retina, one of the highest-oxygen-consuming tissues of the body. We were able to determine whether Ngb a hexacoordinated globin also forms a free radical intermediate when reacting with H2O2.